A highly conserved protein of unknown function in Sinorhizobium meliloti affects sRNA regulation similar to Hfq - PubMed (original) (raw)

. 2011 Jun;39(11):4691-708.

doi: 10.1093/nar/gkr060. Epub 2011 Feb 15.

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A highly conserved protein of unknown function in Sinorhizobium meliloti affects sRNA regulation similar to Hfq

Shree P Pandey et al. Nucleic Acids Res. 2011 Jun.

Abstract

The SMc01113/YbeY protein, belonging to the UPF0054 family, is highly conserved in nearly every bacterium. However, the function of these proteins still remains elusive. Our results show that SMc01113/YbeY proteins share structural similarities with the MID domain of the Argonaute (AGO) proteins, and might similarly bind to a small-RNA (sRNA) seed, making a special interaction with the phosphate on the 5'-side of the seed, suggesting they may form a component of the bacterial sRNA pathway. Indeed, eliminating SMc01113/YbeY expression in Sinorhizobium meliloti produces symbiotic and physiological phenotypes strikingly similar to those of the hfq mutant. Hfq, an RNA chaperone, is central to bacterial sRNA-pathway. We evaluated the expression of 13 target genes in the smc01113 and hfq mutants. Further, we predicted the sRNAs that may potentially target these genes, and evaluated the accumulation of nine sRNAs in WT and smc01113 and hfq mutants. Similar to hfq, smc01113 regulates the accumulation of sRNAs as well as the target mRNAs. AGOs are central components of the eukaryotic sRNA machinery and conceptual parallels between the prokaryotic and eukaryotic sRNA pathways have long been drawn. Our study provides the first line of evidence for such conceptual parallels. Furthermore, our investigation gives insights into the sRNA-mediated regulation of stress adaptation in S. meliloti.

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Figures

Figure 1.

Figure 1.

Sinorhizobium meliloti ORF, SMc01113/ybey, shares similarities with Ago protein. (A) Alignment of the protein sequences of the S. meliloti and E. coli homologs, SMc01113 and YbeY. (B) Alignment of protein sequences of the YbeY and Ago-Mid domain. Structural alignment of Ago-Mid domain (red) and YbeY (green) from N. crassa (C) and A. aeolicus AGO (2NUB) proteins (D). Manually docked PO4 shows potential interactions with the Arg59, K61 residues (E) in the positively charged cavity (F). (G) The lowest energy docked 4mer RNA (Cluspro 2.0 web server) onto YbeY surface fits nicely into the protein cavity, with negatively charged RNA backbone aligned towards the positively charged protein surface.

Figure 2.

Figure 2.

Loss of hfq or SMc01113 makes S. meliloti similarly sensitive to environmental stresses. (A) smc01113 mutant swarms as less efficiently as the hfq mutants than the wild-type strain. Strains were plated on the swarming plates (0.3% agar) and incubated at 30°C. Results of swarming for the three strains were evaluated by measuring colony progression every day for 3 days. (B and C) show sensitivity of the smc01113 and the hfq mutants towards variety of stresses. Strains were grown with or without stress agents as described in the text and OD600 were measured, and percent residual growth (B) after 24 and 48 h (for paraquat, MMS, ethanol and NaCl) or (C) 48 h (for heat shock, SDS and cefotaxime) was calculated.

Figure 3.

Figure 3.

Loss of hfq or SMc01113 similarly down-regulates target transcript accumulation. WT and the smc01113 and the hfq mutants were grown in the RMM till exponential (ppiA, katB and cysK1) or stationary phase (sodC, agpA, livK, ehuB and atpD), RNA was extracted and qPCR was performed. Transcript accumulation in the WT strain was fixed to 1 and relative transcript abundance to WT in the two mutants was evaluated. ‘Single’ and ‘double asterisk’ shows significant difference at P ≤ 0.05 and P ≤ 0.01, respectively.

Figure 4.

Figure 4.

Up-regulated target transcript accumulation in the S. meliloti mutated for hfq or SMc01113. WT and the smc01113 and the hfq mutants were grown in the RMM till stationary phase, RNA was extracted and qPCR was performed. Transcript accumulation in the WT strain was fixed to 1 and relative transcript abundance to WT in the two mutants was evaluated.

Figure 5.

Figure 5.

Loss of hfq or SMc01113 similarly deregulates sRNA accumulation. The WT, smc01113 and hfq mutants, were grown in the RMM till exponential (sras03, 11, 16, 45, 47, 51 and 65) or stationary phase (sra35 and sra63), RNA was extracted and qPCR was performed. Transcript accumulation in the WT strain was fixed to 1 and relative transcript abundance to WT in the two mutants were evaluated as in Figures 3 and 4. ‘Single’ and ‘double asterisk’ shows significant difference at P ≤ 0.05 and P ≤ 0.01, respectively.

Figure 6.

Figure 6.

sRNA-target relation in S. meliloti and conservation of motifs in sRNAs and their possible targets in other related rhizobium species. (A) Correlation between the direction of accumulation of the sRNAs and their target in the S. meliloti strains mutated for hfq and SMc01113, as derived from Figures 3–5. Red and green shows inverse correlation, and yellow shows that the sRNAs and their predicted targets show accumulation in the similar direction. (B) Schematic representation of the three sra16 binding sites in the dppA1 gene. Sra16 was down-regulated whereas dppA1 was up-regulated in the hfq and smc01113 mutants. (C) Conservation of the target-sRNA motif sequences in the three related rhizobium species. Weblogos were generated as described in the text.

Figure 7.

Figure 7.

Over-expression of sRNAs affect accumulation of their predicted targets. (A and B) show the levels of over-expression of sra16 and 35, respectively. (C, E and G) show down-regulation of sra16 targets. (D, F and H) show the changes in putative sra35 targets. Sras 16 and 35 differently regulate the accumulation of common target, glnA (C and D). sRNA-targeting regions were predicted in the coding-region.

Figure 8.

Figure 8.

(A) Directed yeast two hybrid of YbeY. No interaction was detected. (B) Growth of the WT, smc01113 and hfq single, and smc01113 hfq double mutant in LB-MC. Insert shows doubling time of WT and each of the three mutants in LB-MC.

References

    1. LeVier K, Phillips RW, Grippe VK, Roop RM, Walker GC. Similar requirements of a plant symbiont and a mammalian pathogen for prolonged intracellular survival. Science. 2000;287:2492–2493. - PubMed
    1. Becker A, Berges H, Krol E, Bruand C, Ruberg S, Capela D, Lauber E, Meilhoc E, Ampe F, de Bruijn FJ, et al. Global changes in gene expression in Sinorhizobium meliloti 1021 under microoxic and symbiotic conditions. Mol. Plant Microbe Interact. 2004;17:292–303. - PubMed
    1. Gibson KE, Kobayashi H, Walker GC. Molecular determinants of a symbiotic chronic infection. Ann. Rev. Genet. 2008;42:413–441. - PMC - PubMed
    1. Jones KM, Kobayashi H, Davies BW, Taga ME, Walker GC. How rhizobial symbionts invade plants: the Sinorhizobium-Medicago model. Nat. Rev. Microbiol. 2007;5:619–633. - PMC - PubMed
    1. Deakin WJ, Broughton WJ. Symbiotic use of pathogenic strategies: rhizobial protein secretion systems. Nat. Rev. Microbiol. 2009;7:312–320. - PubMed

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